The present invention relates to a histone deacetylase homologue from the hyperthermophilic bacterium Aquifex aeolicus, HDLP (histone deacetylase-like protein; also known as AcuC1), which shares 35.2% sequence identity with human histone deacetylase (HDAC1), that can be co-crystallized with an inhibitory ligand, and more particularly, to the detailed crystallographic data obtained from said co-crystallization which is disclosed herein. The invention also relates to methods of using the crystal structure and x-ray crystallographic coordinates of the apo-HDLP inhibitor-bound HDLP to design, isolate and screen compounds which bind to and inhibit the active site of HDLP and HDLP-related proteins, such as those proteins belonging to the HDAC family, including HDAC1.
The reversible modification of histones by-acetylation is associated with changes in nucleosome conformation and chromatin structure, and plays an important role in the regulation of gene expression (reviewed in Davie and Chadee, 1998, J. Cell Biochem. Suppl. 30–31:203–213). The histone acetylase and deacetylase enzymes that carry out these modifications are involved in many cellular processes such as cell cycle progression and differentiation, and their deregulation is associated with several types of human cancer (reviewed in Kouzarides, 1999, Curr. Opin. Genet. Dev. 9:40–48; Hassig et al., 1997, Chem. Biol. 4:783–789; Fenrick and Heibert, 1998, J. Cell. Biochem. Suppl. 30–31:194–202).
Recently, several experimental-antitumor compounds, such as trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid (SAHA), and phenylbutyrate have been shown to act, at least in part, by inhibiting histone deacetylases. Richon et al., 1998, Proc. Natl. Acad. Sci., USA 95:3003–3007; Yoshida et al., 1990, J. Biol. Chem. 265:17174–17179; Kijima et al., 1993, J. Biol. Chem. 268:22429–22435. Additionally, diallyl sulfide and related molecules (Lea et al., 1999, Int. J. Oncol. 2:347–352), oxamflatin (Kim et al., 1999, Oncogene 15:2461–2470), MS-27–275, a synthetic benzamide derivative (Saito et al., 1999, Proc. Natl. Acad. Sci. 96:4592–4597), butarate derivatives (Lea and Tulsyan, 1995, Anticancer Res. 15:879–883), FR901228 (Nokajima et al., 1998, Exp. Cell Res. 241:126–133), depudecin (Kwon et al., 1998, Proc. Natl. Acad. Sci. USA 95:3356–3361), and m-carboxysinnamic acid bishydroxamide (CBHA; Richon et al., Proc. Natl. Acad. Sci. USA 95:3003–3007) have been shown to inhibit histone deacetylases. In vitro, these compounds can inhibit the growth of fibroblast cells by causing cell cycle arrest in the G1 and G2 phases (Richon et al., 1996, Proc. Natl. Acad. Sci. USA 93:5705–5708; Kim et al., 1999, Oncogene 18:2461–2470; Yoshida et al., 1995, Bioessays 17:423–430; Yoshida & Beppu, 1988, Exp. Cell. Res. 177:122–131), and can lead to the terminal differentiation and loss of transforming potential of a variety of transformed cell lines. Richon et al., 1996, Proc. Natl. Acad. Sci. USA 93:5705–5708; Kim et al., 1999, Oncogene 18:2461–2470; Yoshida et al., 1987, Cancer Res. 47:3688–3691. In vivo, phenylbutyrate is effective in the treatment of acute promyelocytic leukemia in conjunction with retinoic acid. Warrell et al., 1998, J. Natl. Cancer Inst. 90:1621–1625. SAHA is effective in preventing the formation of mammary tumors in rats, and lung tumors in mice. Desai et al., 1999, Proc. AACR 40: abstract #2396; Cohen et al., Cancer Res., submitted.
Histone deacetylases catalyze the removal of acetyl groups from the ∈-amino groups of lysine residues clustered near the N-terminus of nucleosomal histones, and this process is associated with transcriptional repression (reviewed in Struhl, 1998, Genes Dev. 12:599–606). Deletion of the yeast histone deacetylase gene, rpd3, or its pharmacological inactivation with trichostatin A reduces the transcriptional repression in a subset of promoters, such as those of Ume6-regulated genes. Kadosh & Struhl, 1998, Mol. Cell. Biol. 18:5121–5127. This is accompanied by the increased acetylatibn of H4 histones in the repressed promoter and its vicinity, but has no effect on histones at promoter distal regions. Kadosh & Struhl, 1998, Mol. Cell. Biol. 18:5121–5127; Rundlett et al., 1998, Nature 392:831–835.
Histone deacetylases are recruited to specific promoters by associating with DNA-binding transcriptional repressors, either directly or through co-repressors that bridge the deacetylase to the transcriptional repressors. For example, the Mad and Ume6-repressors bind to the co-repressor Sin3A (Laherty et al., 1997, Cell 89.349–356; Hassig et al., 1997, Cell 89:341–347; Kadosh & Struhl, 1997, Cell 89:365–371), and the nuclear receptors bind N-CoR and the related SMRT co-repressors. Nagy et al., 1997, Cell 89:373–380; Alland et al, 1997, Nature 387:49–55; Heinzel et al, 1997, Nature 20 387:43–48.
The deregulation of histone deacetylase recruitment appears to be one of the mechanisms through which these enzymes contribute to tumorigenesis. In acute promyelocytic leukemia (APL), chromosomal translocations fuse the retinoic acid receptor-α (RARα) to either PLZF or to PML. These fusion oncoproteins have aberrant transcriptional repression activity resulting, in part, through the recruitment of a co-repressor and, in turn, HDACs. Grignani et al, 1998, Nature 391:815–818; Lin et al., 1998, Nature 391:811–814. Treatment of PLZF-RARα APL cells with TSA enhances their responsiveness to retinoic acid-induced differentiation. Grignani et al, 1998, Nature 391:815–818; Lin et al., 1998, Nature 391:811–814.
The histone deacetylases comprise a large family of proteins, conserved from yeast to man, and are divided into two related classes. Class I is characterized by human HDACI, 2, 3 (Taunton et al., 1996, Science 272:408–411; Yang et al., 1996, Proc. Natl. Acad. Sci. USA 93:12845–12850; Emiliani et al., 1998, Proc. Natl. Acad. Sci. USA 95:2795–2800), and yeast RPD3 (Videl & Gaber, 1991, Mol. Cell. Biol. 11:6317–6327), and class II by the human HDAC4, 5, 6 (Grozinger et al., 1999, Proc. Natl. Acad. Sci. USA 96 :4868–4873; Fischle, et al., 1999, J. Biol. Chem. 274:11713–11720), and yeast HDA1 (Rundlett et al., 1996, Proc. Natl. Acad. Sci. USA 93:14503–14508). The two classes share a −390 amino acid region of sequence similarity, comprising the deacetylase core, but are divergent outside this region. The histone deacetylase genes belong to an even larger superfamily (Leipe & Landsman, 1997, Nucleic Acids Res. 25:3693–3697) that contains the prokaryotic acetoin utilization proteins (AcuC; 28.1%, sequence identity to HDAC1), and the prokaryotic acetylpolyamine amidohydrolases (APAH; 15.0% sequence identity to HDAC1). The enzymatic activity of AcuC is not clear, but its disruption reduces the ability of B. subtilis to breakdown acetoin and utilize it as a carbon source. Grundy et al., 1993, Mol. Microbiol. 13:259–271. APAHs catalyze the deacetylation of polyamines by cleaving a non-peptide amide bond (reviewed in Leipe & Landsman, 1997, Nucleic Acids Res. 25:3693–3697).
It is useful to address the questions of how HDACs and HDAC-related proteins catalyze the deacetylation of histones and how the above-referenced compounds, particularly those compounds with antitumor activity, inhibit this activity in order to better understand the mechanism of inhibition of HDACs and to facilitate discovery of additional useful compounds which may inhibit this activity. To this end, the present invention has determined the three dimensional structure of a HDAC1-like protein from the thermophilic bacterium Aquifex aeolicus, herein after HDLP. The determination, of the nucleic acid coding sequence of HDLP was described by Deckert et al., 1998, Nature 392:353–358. The encoded 375 residue protein, whose sequence was determined from the nucleic acid encoding sequence, shares 35.2% amino acid sequence identity with HDAC1, deacetylates histones in vitro, and is inhibited by TSA, SAHA and several other HDAC inhibitors. The determination of the three-dimensional structure of HDLP is useful in the design, identification and screening of new HDAC family inhibitory compounds which are useful for the inhibition of cell growth both in vivo and in vitro.